Objective:

The goal of today’s lab is to use to different types of DNA extraction methods. We will use the MoBio Power Soil Kit and the Chelex Extraction protocol to determine which procedure would work best when we use the DNA in later experiments. The main go is to try these new procedures because the one’s used in the past did not yield any significant results.

Purpose:

Due to the fact that we are implementing new procedures, we may be able to determine sources of error if any arise since we are using different protocols. We are trying new protocols to compare their results which will help us to determine whether or not our metabarcoding goal could be achieved. We also split up the protocols within our table groups which would also help us to interact with more people and discuss the protocols we are each using.

Procedure:

MoBio/Power Soil Kit-

1.) Weigh out 0.3 grams of soil and add it to the powerbead tubes then gently vortex.

2.) Add 60 micro-liters of solution C1 and invert several times or vortex briefly to mix.

3.) Place the powerbead tube horizontally on a flat-bed vortex pad with tape and vortex at maximum fro ten minutes.

4.) Centrifuge tubes at 10,000 x gravity for thirty seconds at room temperature then transfer the supernatant to a clean 2mL collection tube.

5.) Add 250 micro-liters of solution C2 and vortex briefly.

6.) Incubate for five minutes at 4 degrees Celsius (place in the refrigerator).

7.) Centrifuge the tube at room temperature for one minute at 10,000 x gravity.

8.) Then transfer 600 micro-liters of supernatant to a clean 2mL collection tube.

9.) Add 200 micro-liters of solution C3 and vortex briefly then incubate at 4 degrees Celsius by putting the tube in the green tube rack within the refrigerator.

10.) Centrifuge the tubes at the room temperature for one minute at 10,000 x gravity.

11.) Transfer up to 750 micro-liters of supernatant to a clean 2mL collection tube.

12.) Add 1.2mL of solution C4 to the supernatant and vortex for five seconds.

13.) Load about 675 micro-liters onto a spin filter and centrifuge at 10,000 x gravity for one minute at room temperature. Discard the flow through and add 675 micro- liters of supernatant to the spin filter and centrifuge again at room temperature for one minute.

14.) Load the remaining supernatant onto the spin filter and centrifuge at 10,000 x gravity for one minute at room temperature.

15.) Add 500 micro-liters of C5 solution and centrifuge at room temperature for thirty seconds at 10,000 x gravity.

16.) Discard the flow through from the 2mL collection tube

17.) Centrifuge at room temperature for one minute at 10,000 x gravity.

18.) Carefully place the spin filter into a clean 2mL collection tube, then add 100 micro-liters of solution C6 to the center of the white filter membrane.

19.) Centrifuge at room temperature for thirty seconds at 10,000 x gravity.

20.) Discard the spin filter and store in the freezer.

Chelex Extraction-

1.) Transfer 300-500 micro-liters of ciliate culture from a non-flooded plate to a microcentrifuge tube. Record which ciliate culture you are extracting and label your tubes.

2.) Centrifuge at 6,000 x gravity for five minutes, after centrifugation, using a pipet to extract out the supernatant.

3.) Weigh 0.5 grams of chelex and transfer to a 15mL conical tube then add D.I. water to the 10mL line of the tube.

4.) Add 200 micro-liters of 5% Chelex to the pellet in the microcentrifuge tube, then vortex for one minute (use a p1000 micropipette and cut the tip slightly to allow more of the sample through and avoid clogging).

5.) Incubate for 30 minutes in a 56 degree Celsius water bath to help break open the cells and denature some of the proteins.

6.) Then boil for eight minutes in a 100 degree Celsius water bath.

7.) Vortex for one minute.

8.) Centrifuge at 16,000 x gravity for three minutes (this is to pellet cellular debris and chelex beads).

9.) Use a micropipette to transfer the supernatant with DNA in solution to a clean microcentrifuge tube (try to avoid pipetting out the Chelex beads).

10.) Make sure to label the top and sides of the microcentrifuge tubes.

Controls-

*Create a control by using the paramecium sample and performing the procedures identically to before. this will show will help us determine wether or not the cells are destroyed further or the protocols we followed did not produce the results we wanted since we know that the control would infect have cells.

Data/Observations:

In this lab, the soil from the non- flooded plate in the Chelex procedure looked a lot lighter compared to the soil used in the MoBio kit. I think this had much to do with the fact that the PowerSoil procedure called for 0.3 grams of actual soil where as the other procedure try to extract cells from the water of a non-flooded soil sample plate. The PowerSoil would probably be a more pure procedure do to the fact that we used the exact soil we wanted to isolate versus trying to extract ciliates from a well plate. I thought it was interesting that we placed the soil directly into the power bead tube because we had always previously extracted our cells from some time of aqueous environment. It was also interesting when comparing the two procedures because they were very different, yet supposed to yield the same result. The MoBio kit incubated at fairly cold temperatures wheres the Chelex protocol incubated at extremely hot temperatures. I am curious to see if each samples will yield the same results in gel electrophoresis or using the nano drop technology as our experiment progresses.

Storage:

In both protocols, we stored the samples in the freezer in a tube rack for later use. This will help preserve our DNA so that we may be able to continue to use these samples the next time we are in lab.

Future Goals:

In the future I hope that we will be able to perform PCR and eventually have a successful run of gel electrophoresis where bands of DNA are present. I hope that using these new kits for DNA extraction with help us to further are original goal of metabarcoding ciliates within the soil.